Pharmaceutical compositions directed to Erb-B1 receptors

ABSTRACT

The invention relates to pharmaceutical compositions comprising different molecules, preferably monoclonal antibodies, each comprising epitopes that bind simultaneously to different sites within the same ErbB receptor domain, preferably the ErbB1 receptor domain. The preferred antibodies according to this invention are MAb 425 and MAb 225 each in its murine, chimeric and humanized version. The invention relates to the use and methods for an improved treatment of preferably tumors by means of said compositions.

FIELD OF THE INVENTION

The invention relates to pharmaceutical compositions comprisingdifferent biological molecules, preferably monoclonal antibodies, eachcomprising epitopes that bind simultaneously to different epitopes ofthe same ErbB receptor domain, especially ErbB1 receptor domain. Thepreferred antibodies according to this invention are MAb 425 and MAb 225each in its murine, chimeric and humanized version. The inventionrelates to the use and methods for an improved treatment of preferablytumors by means of said compositions.

BACKGROUND OF THE INVENTION

Biological molecules, such as monoclonal antibodies (MAbs) or otherproteins/polypeptides, as well as small chemical compounds directedagainst various receptors and other antigens on the surface of tumorcells are known to be suitable for tumor therapy for more than twentyyears. With respect to the antibody approach, most of these MAbs arechimerized or humanized to improve tolerability with the human immunesystem. MAbs or above-mentioned chemical entities specifically bind totheir target structures on tumor cells and in most cases also on normaltissues and can cause different effects that dependent on their epitopespecificity and/or functional characteristics of the particular antigen.MAbs to orphan receptors or other non-functional cell surface moleculesas well as MAbs against structures outside the ligand-binding site offunctionally active receptors (e.g. growth factor receptors with kinaseactivity) would be expected to induce primarily immune effectorfunctions against the target cell (antibody-dependent cell-mediatedcytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)).Additionally, depending on the properties of antigen and MAb, binding ofthe antibody can result in cross-linking of the receptors. Consequentinternalization of the receptor-antibody complexes may result in aprolonged down-modulation of the receptor density on the cell surface.

MAbs or small chemical compounds that bind to an epitope within theligand-binding site or in its direct neighborhood compete for binding ofnatural ligands to their receptor and thus reduce or completely inhibitligand binding and can displace already bound ligands from theirreceptors. This receptor blockade inhibits ligand-dependent receptoractivation and downstream signaling. For example, blockade of ErbBreceptors, such as the epidermal growth factor receptor (EGFR), bymonoclonal antibodies results in various cellular effects includinginhibition of DNA synthesis and proliferation, induction of cell cyclearrest and apoptosis as well as antimetastatic and antiangiogeneticeffects.

ErbB receptors are typical receptor tyrosine kinases that wereimplicated in cancer in the 1980s. Tyrosine kinases are a class ofenzymes that catalyze the transfer of the terminal phosphate ofadenosine triphosphate to tyrosine residues in protein substrates.Tyrosine kinases are believed, by way of substrate phosphorylation, toplay critical roles in signal transduction for a number of cellfunctions. Though the exact mechanisms of signal transduction is stillunclear, tyrosine kinases have been shown to be important contributingfactors in cell proliferation, carcinogenesis and cell differentiation.Tyrosine kinases can be categorized as receptor type or non-receptortype. Both receptor-type and non-receptor type tyrosine kinases areimplicated in cellular signaling pathways leading to numerous pathogenicconditions, including cancer, psoriasis and hyperimmune responses. Manytyrosine kinases are involved in cell growth as well as in angiogenesis.The non-receptor type of tyrosine kinases is also comprised of numeroussubfamilies, including Src, Frk, Btk, Csk, Abl, Zap70, Fes/Fps, Fak,Jak, Ack, and LIMK. Each of these subfamilies is further sub-dividedinto varying receptors. For example, the Src subfamily is one of thelargest and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr, and Yrk.The Src subfamily of enzymes has been linked to oncogenesis. For a moredetailed discussion of the non-receptor type of tyrosine kinases, seeBolen Oncogene, 8:2025-2031 (1993).

Receptor type tyrosine kinases have an extracellular, a transmembrane,and an intracellular portion, while non-receptor type tyrosine kinasesare wholly intracellular. Receptor-linked tyrosine kinases aretransmembrane proteins that contain an extracellular ligand bindingdomain, a transmembrane sequence, and a cytoplasmic tyrosine kinasedomain. The receptor-type tyrosine kinases are comprised of a largenumber of transmembrane receptors with diverse biological activity.

Different subfamilies of receptor-type tyrosine kinases have beenidentified. Implicated tyrosine kinases include fibroblast growth factor(FGF) receptors, epidermal growth factor (EGF) receptors of the ErbBmajor class family, and platelet-derived growth factor (PDGF) receptors.Also implicated are nerve growth Factor (NGF) receptors, brain-derivedneurotrophic Factor (BDNF) receptors, and neurotrophin-3 (NT-3)receptors, and neurotrophin-4 (NT-4) receptors.

One receptor type tyrosine kinase subfamily, designated as HER or ErbBsubfamily, is comprised of EGFR (ErbB1), HER2 (ErbB2 or p185neu), HER3(ErbB3), and HER4 (ErbB4). Ligands of this subfamily of receptorsinclude epithelial growth factor (EGF), TGF-α, amphiregulin, HB-EGF,betacellulin, heregulin and neuregulins. The PDGF subfamily includes theFLK family which is comprised of the kinase insert domain receptor(KDR).

EGFR, encoded by the erbB1 gene, has been causally implicated in humanmalignancy. In particular, increased expression of EGFR has beenobserved in breast, bladder, lung, head, neck and stomach cancer as wellas glioblastomas. Increased EGFR receptor expression is often associatedwith increased production of the EGFR ligand, transforming growth factoralpha (TGF-a), by the same tumor cells resulting in receptor activationby an autocrine stimulatory pathway (Baselga and Mendelsohn, Pharmac.Ther. 64:127-154 (1994)).

The EGF receptor is a transmembrane glycoprotein which has a molecularweight of 170.000, and is found on many epithelial cell types. It isactivated by at least three ligands, EGF, TGF-α (transforming growthfactor alpha) and amphiregulin. Both epidermal growth factor (EGF) andtransforming growth factor-alpha (TGF-a) have been demonstrated to bindto EGF receptor and to lead to cellular proliferation and tumor growth.These growth factors do not bind to HER2 (Ulrich and Schlesinger, 1990,Cell 61, 203). In contrary to several families of growth factors, whichinduce receptor dimerization by virtue of their dimeric nature (e.g.PDGF) monomeric growth factors, such as EGF, contain two binding sitesfor their receptors and, therefore, could principally cross-link twoneighboring EGF receptors (Lemmon et al., 1997, EMBO J. 16, 281). Recentstudies (J. Schlessinger, 2002, Cell 110, 669) show that receptordimerization is mediated by receptor-receptor interactions in which aloop protruding from neighboring receptors mediates receptordimerization and activation.

Receptor dimerization is essential for stimulating of the intrinsiccatalytic activity and for the self-phosphorylation of growth factorreceptors on tyrosine residues. The latter serve as docking sites forvarious adaptor proteins or enzymes, which simultaneously initiate manysignaling cascades. In higher eukaryotes, the simple linear pathway hasevolved into a richly interactive, multi-layered network in whichcombinatorial expression and activation of components permitscontext-specific biological responses throughout development. The ErbBnetwork might integrate not only its own inputs but also heterologoussignals, including hormones, lymphokines, neurotransmitters and stressinducers.

It should be remarked that receptor protein tyrosine kinases (PTKs) areable to undergo both homo- and heterodimerization, wherein homodimericreceptor combinations are less mitogenic and transforming (no or weakinitiation of signaling) than the corresponding heterodimericcombinations. Heterodimers containing ErbB2 are the most potentcomplexes (see review articles by Yarden and Sliwkowski, 2001, NatureReviews, Molecular cell Biology, volume 2, 127-137; Tzahar and Yarden,1998, BBA 1377, M25-M37).

It has been demonstrated that anti-EGF receptor antibodies whileblocking EGF and TGF-α binding to the receptor appear to inhibit tumorcell proliferation. In view of these findings, a number of murine andrat monoclonal antibodies against EGF receptor have been developed andtested for their ability inhibit the growth of tumor cells in vitro andin vivo (Modjtahedi and Dean, 1994, J. Oncology 4, 277). Humanizedmonoclonal antibody 425 (hMAb 425, U.S. Pat. No. 5,558,864; EP 0531 472)and chimeric monoclonal antibody 225 (cMAb 225), both directed to theEGF receptor, have shown their efficacy in clinical trials. The C225antibody (Cetuximab) was demonstrated to inhibit EGF-mediated tumor cellgrowth in vitro and to inhibit human tumor formation in vivo in nudemice. The antibody as well as in general all anti-EGFR antibodies,appears to act, above all, in synergy with certain chemotherapeuticagents (i.e., doxorubicin, adriamycin, taxol, and cisplatin) toeradicate human tumors in vivo in xenograft mouse models (see, forexample, EP 0667165). Ye et al. (1999, Oncogene 18, 731) have reportedthat human ovarian cancer cells can be treated successfully with acombination of both chimeric MAb 225 and humanized MAb 4D5 which isdirected to the HER2 receptor.

The second member of the ErbB family, HER2 (ErbB2 or p185neu), wasoriginally identified as the product of the transforming gene fromneuroblastomas of chemically treated rats. The activated form of the neuproto-oncogene results from a point mutation (valine to glutamic acid)in the transmembrane region of the encoded protein. Amplification of thehuman homologue of neu is observed in breast and ovarian cancers andcorrelates with a poor prognosis (Slamon et al., Science, 235: 177-182(1987); Slamon et al., Science, 244:707-7 12 (1989); U.S. Pat. No.4,968,603). ErbB2 (HER2) has a molecular weight of about 185.000, withconsiderable homology to the EGF receptor (HER1), although a specificligand for HER2 has not yet been clearly identified so far. The antibody4D5 directed to the HER2 receptor, was further found to sensitizeErbB2-overexpressing breast tumor cell lines to the cytotoxic effects ofTNFα (U.S. Pat. No. 5,677,171). A recombinant humanized version of themurine anti-ErbB2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2 or HERCEPTIN®;U.S. Pat. No. 5,821,337) is clinically active in patients withErbB2-overexpressing metastatic breast cancers that have receivedextensive prior anti-cancer therapy (Baselga et al., J. Clin. Oncol.14:737-744 (1996)). HERCEPTIN® received marketing approval in 1998 forthe treatment of patients with metastatic breast cancer whose tumorsoverexpress the ErbB2 protein.

Besides anti-ErbB antibodies there are numerous small chemical moleculeswhich are known to be potent inhibitors of ErbB receptor moleculesblocking the binding site of the natural ligands (see detaileddescription), or blocking the tyrosine residues of the binding site ofthe receptor kinase, thus preventing phosphorylation and further cascadesignaling. One representative showing high efficacy in clinical trialsis Iressa™ (ZD-1839) which can be applied for NSCLC indication(non-small cell lung cancer).

Although there are already some promising drugs and methods of treatmenttumors under development and in the market, there is a continuous needfor further agents and pharmaceutical compositions and combinations withimproved properties and enhanced efficacy.

SUMMARY OF THE INVENTION

The invention is based on the observation of the inventors, that certainreceptor tyrosine kinases such as ErbB receptor antibody molecules,preferably ErbB1 receptor (EGFR) antibody molecules, which areoverexpressed on diseased cell surfaces, e.g. tumor cells, have specificepitope sites within the natural ligand binding domain to whichsimultaneously different antibody molecules may be bound without or onlynegligible mutual hindrance. Evidently, these antibody molecules possessbinding epitopes which are with respect to their three-dimensionalconfiguration relatively small, as compared with the total size of thebinding domain of the receptor molecule. They induce an increasedmodulation activity of pathway signaling, preferably an increasedblocking of the ErbB receptor and, thus, of the complete signalingcascade. The present invention describes for the first time the newconcept in tumor therapy to administer to an individual one or morebiologically and therapeutically effective agents that block or inhibitan ErbB receptor, preferably the EGF receptor (EGFR) (ErbB1), by bindingsaid agent(s) to at least a first and a second different epitope,preferably within the natural ligand domain of the same receptor. Itcould be found that, e.g., two or more, preferably two, distinctreceptor-antagonistic molecules can bind simultaneously to the samereceptor domain, preferably of the same receptor molecule, withoutmutual hindrance or competition, thus enabling a higher density ofantagonist bound to the receptor and affecting (by a less ability tobind natural (agonistic) ligands such as EGF or TGF a) a much strongerinhibition of the signaling cascade of the corresponding receptormolecules as monomeric or dimeric units. This should lead to a strongerinhibition of tumor growth and/or increased apoptosis of solid tumors ortumor metastases. Said molecules may be small chemical and syntheticcompounds or proteins, polypeptides or peptides, immunoglobulins, suchas antibodies or fragments thereof, or immunoconjugates. Preferredmolecules are anti-ErbB antibodies, especially anti-EGFR and anti-Her2antibodies as specified above and below, and fragments thereof,preferably F(ab′)2. In a preferred embodiment of this invention twodifferent anti-EGFR antibodies are administered to an individual,preferably MAb 425 in a humanized, chimeric or murine version or afragment thereof, such as a F(ab′)2, and MAb 225, in a humanized,chimeric or murine version or a fragment thereof, such as a F(ab′)2.Most preferred is the combinatorial application of humanized MAb 425 andchimeric MAb 225 as a whole antibody or as F(ab′)2 fragment.

It is however also possible to use relatively short synthetic(poly)peptides deriving and produced from said antibody constructscomprising amino acid sequences of one, two or three CDRs of therespective antibody, wherein, optionally, in order to increase thebinding affinity and/or avidity to the receptor, some amino acids withinthe antigen binding site or in close vicinity thereof (1-5 amino acids)may be modified by preferably substitution. Such synthetic peptideswhich can bind to the receptor in a comparable manner as the respectiveantibodies have the advantage of a simple and cheaper way ofmanufacture. It is also possible to synthesize one single peptide thatcomprises said CDR-derived amino acid sequences of the first molecule aswell as of the second molecule, for example a (poly)peptide comprisingamino acid sequences deriving from 1-3 CDRs of MAb 425 and 1-3 CDRs ofMAb 225.

It was found that the pharmaceutical compositions according to thisinvention can affect enhanced cross-linking/dimerization of different oridentical ErbB receptors, enhanced blocking/inhibition of ErbBreceptors, and enhanced induction of modulation of ErbBreceptor-specific pathway signaling as compared with a single moleculecomprising one of said binding sites only. In other words: a mixture of,for example, MAb 425 and MAb 225 elicit an enhanced inhibition anddown-regulation of EGFR as compared to MAbs 425 or 225 applied as singleagent in the same concentration.

Although above-described observations were made for ErbB receptors astarget receptor molecules only it should be pointed out that thescientific principle discovered by the inventors and stated out aboveand below might be also applicable for other biological receptors otherthan ErbB.

Optionally, the composition according to this invention comprisesfurther therapeutically active compounds which may support and enhancethe efficacy of above-said molecules. Such agents may cytotoxic agentsand preferably antagonistic molecules, such as tyrosine kinaseantagonists, other ErbB antagonists, hormone growth receptorantagonists, protein kinase antagonists, anti-angiogenic agents, orcytokines. Such molecules usable in the present invention are specifiedin more detail below.

According to this invention the therapeutically active agents may alsobe provided by means of a pharmaceutical kit comprising a packagescontaining one or more of said antagonistic agents in single or separatecontainers. The therapy with this combinations may include optionallytreatment with radiation. Principally, the administration can beaccompanied by radiation therapy, wherein radiation treatment can bedone substantially concurrently or before or after the drugadministration. The administration of the different agents of thecombination therapy according to the invention can also be achievedsubstantially concurrently or sequentially. Tumors, bearing receptors ontheir cell surfaces involved in the development of the blood vessels ofthe tumor, may be successfully treated by the combination therapy ofthis invention.

It is known that tumors elicit alternative routes for their developmentand growth. If one route is blocked they often have the capability toswitch to another route by expressing and using other receptors andsignaling pathways. Therefore, the pharmaceutical combinations of thepresent invention may block several of such possible developmentstrategies of the tumor and provide consequently various benefits. Thecombinations according to the present invention are useful in treatingand preventing tumors, tumor-like and neoplasia disorders and tumormetastases, which develop and grow by activation of their relevanthormone receptors which are present on the surface of the tumor cells.Preferably, the different combined agents of the present invention areadministered in combination at a low dose, that is, at a dose lower thanhas been conventionally used in clinical situations. A benefit oflowering the dose of the compounds, compositions, agents and therapiesof the present invention administered to an individual includes adecrease in the incidence of adverse effects associated with higherdosages. For example, by the lowering the dosage of an agent describedabove and below, a reduction in the frequency and the severity of nauseaand vomiting will result when compared to that observed at higherdosages. By lowering the incidence of adverse effects, an improvement inthe quality of life of a cancer patient is contemplated. Furtherbenefits of lowering the incidence of adverse effects include animprovement in patient compliance, a reduction in the number ofhospitalizations needed for the treatment of adverse effects, and areduction in the administration of analgesic agents needed to treat painassociated with the adverse effects. Alternatively, the methods andcombination of the present invention can also maximize the therapeuticeffect at higher doses.

The combinations according to the inventions show an astonishingsynergetic effect. In administering the combination of drugs real tumorshrinking and disintegration could be observed during clinical studieswhile no significant adverse drug reactions were detectable.

In detail the invention refers to:

-   -   A pharmaceutical composition comprising one or more biologically        and/or therapeutically effective antibody molecules (or a        fragment thereof) having the ability to bind to different        epitopes of a binding domain of an ErbB receptor molecule,        wherein said one or more antibody molecule(s) comprise(s) at        least a binding site that binds to a first specific epitope of        said receptor binding domain and at least another binding site        that binds to a second specific epitope of the same ErbB        receptor binding domain.    -   A corresponding pharmaceutical composition, comprising two or        more antibody molecules, wherein one antibody molecule comprises        at least a binding site that binds to a first specific epitope        of said receptor domain and at least another antibody molecule        comprises at least another binding site that binds to a second        specific epitope of the same receptor binding domain.    -   A corresponding pharmaceutical composition, wherein at least one        of said antibody molecules binds to an epitope within the        receptor binding domain to which the natural ligand of the        receptor binds.    -   A corresponding pharmaceutical composition affecting enhanced        blocking and/or inhibition of ErbB receptor, and enhanced        induction of modulation of ErbB receptor-specific pathway        signaling as compared with a single molecule comprising one of        said binding sites only.    -   A corresponding pharmaceutical composition, affecting enhanced        induction of crosslinking and/or dimerization of different        receptor molecules having the same or different specificity.    -   A corresponding pharmaceutical composition, wherein said ErbB        receptor is EGF receptor (EGFR).    -   A corresponding pharmaceutical composition, comprising a first        and a second monoclonal antibody or a biologically active        fragment thereof, each directed to different epitopes of the EGF        receptor.    -   A corresponding pharmaceutical composition, wherein the first        antibody is murine, chimeric or humanized MAb 425.    -   A corresponding pharmaceutical composition, wherein the second        antibody is murine, chimeric or humanized MAb 225.    -   A corresponding pharmaceutical composition, wherein said first        antibody is humanized MAb 425 (h425) and said second antibody is        chimeric MAb 225 (c225, Cetuximab).    -   A corresponding pharmaceutical composition, comprising        additionally a cytotoxic drug.    -   A pharmaceutical kit comprising        -   (i) a first package comprising at least a first biologically            active antibody molecule, or a fragment thereof, that binds            to a first specific epitope of a binding domain of an ErbB            receptor molecule, and (ii) a second package comprising at            least a second antibody molecule, or a fragment thereof,            that binds to a different second specific epitope of the            binding domain of the same ErbB receptor molecule.    -   A corresponding pharmaceutical kit, wherein at least one of said        antibody molecules binds to an epitope within the receptor        binding domain to which the natural ligand of the receptor        binds.    -   A corresponding pharmaceutical kit, wherein said first antibody        molecule is murine, chimeric or humanized monoclonal antibody        425 or a biologically active fragment thereof, and said second        antibody molecule is murine, chimeric or humanized monoclonal        antibody 225 or a biologically active fragment thereof.    -   A corresponding pharmaceutical kit comprising a first package        that comprises humanized MAb 425 (h425) and a second package        that comprises chimeric MAb 225 (c225).    -   A corresponding pharmaceutical kit comprising additionally a        third package comprising a further drug having the ability to        increase the efficacy of the drugs provided by the first and        second package.    -   A corresponding pharmaceutical kit, wherein said additional drug        is a cytotoxic drug.    -   A method for treating tumor related diseases in a patient        comprising administering to said patient a therapeutically        effective amount of (i) at least a first antibody molecule (or a        fragment thereof) that binds to a first specific epitope of a        binding domain of an ErbB receptor molecule and (ii) at least a        second antibody molecule that binds to a different second        specific epitope of the binding region of the same ErbB receptor        molecule.    -   A corresponding method, wherein at least one of said antibody        molecules binds to an epitope within the receptor binding domain        to which the natural ligand of the receptor binds.    -   A corresponding method, wherein said antibody molecule that        binds to said epitope blocks and/or inhibits the receptor, thus        inducing modulation of receptor-specific pathway signaling.    -   A corresponding method, wherein the binding of said antibody        molecule induces crosslinking and/or dimerization of different        receptor molecules having the same or different specificity.    -   A corresponding method, wherein said first antibody molecule is        humanized monoclonal antibody 425 (h425) or a biologically        active fragment thereof, and said second molecule is chimeric or        monoclonal antibody 225 (c225) or a biologically active fragment        thereof.

In a preferred embodiment of the invention the ErbB receptor is the EGFreceptor (EGFR) and the antibodies directed to different epitopes onthis receptor are anti-EGFR antibodies.

Thus, the invention relates in detail to:

-   -   A pharmaceutical composition comprising a first and a second        antibody molecule, or a portion thereof, having the capability        to bind to different epitopes located on same or different ErbB        receptor molecule types, wherein said first antibody molecule or        a portion thereof, comprises binding sites that bind to a first        specific epitope on the ErbB1 receptor molecule type, and said        second antibody molecule comprises binding sites that bind to a        second specific epitope on the same ErbB1 receptor molecule        type.    -   A pharmaceutical composition, wherein at least said first or        said second epitope on the ErbB1 receptor molecule type is        located within the ErbB1 receptor binding domain.    -   A pharmaceutical composition, wherein said first and said second        epitope on the ErbB1 receptor molecule type is located within        the ErbB1 receptor binding domain.    -   A pharmaceutical composition, wherein said receptor binding        domain is the binding domain of the natural ligand of said ErbB1        receptor molecule type.    -   A pharmaceutical composition, wherein the first and second        antibody, or fragment thereof, binds to different epitopes        within the binding domain of the natural ligand(s) of said ErbB1        receptor molecule type.    -   A pharmaceutical composition, wherein blocking and/or inhibition        of the ErbB receptor, and induction of down-regulation of ErbB        receptor-specific pathway signaling is enhanced as compared with        a composition comprising a single antibody molecule which binds        to said first or said second epitope on said ErbB1 receptor        molecule type only.    -   A pharmaceutical composition, wherein the induction of        cross-linking and/or dimerization of ErbB receptor molecules of        the same or different specificity is enhanced as compared with a        composition comprising a single antibody molecule which binds to        said first or said second epitope on said ErbB1 receptor        molecule type only.    -   A pharmaceutical composition, wherein said ErbB receptor        molecules, are involved in cross-linking and/or dimerization,        and are selected from ErbB1 and ErbB2 (Her-2).    -   A pharmaceutical composition, wherein said first and/or said        second antibodies is a monospecific antibody.    -   A pharmaceutical composition, wherein the first antibody is        murine, chimeric or humanized MAb 425.    -   A pharmaceutical composition, wherein the second antibody is        murine, chimeric or humanized MAb 225.    -   A pharmaceutical composition, wherein said first antibody is        humanized MAb 425 (h425) and said second antibody is chimeric        MAb 225 (c225).    -   A pharmaceutical composition according to any of the following        12 pharmaceutical compositions, comprising additionally a        cytotoxic agent:    -   (a) A pharmaceutical composition comprising a first and a second        antibody molecule, or a portion thereof, having the capability        to bind to different epitopes located on same or different ErbB        receptor molecule types, wherein said first antibody molecule or        a portion thereof, comprises binding sites that bind to a first        specific epitope on the ErbB1 receptor molecule type, and said        second antibody molecule comprises binding sites that bind to a        second specific epitope on the same ErbB1 receptor molecule        type.    -   (b) A pharmaceutical composition, wherein at least said first or        said second epitope on the BrbB1 receptor molecule type is        located within the ErbB1 receptor binding domain.    -   (c) A pharmaceutical composition, wherein said first and said        second epitope on the ErbB1 receptor molecule type is located        within the ErbB1 receptor binding domain.    -   (d) A pharmaceutical composition, wherein said receptor binding        domain is the binding domain of the natural ligand of said ErbB1        receptor molecule type.    -   (e) A pharmaceutical composition, wherein the first and second        antibody, or fragment thereof, binds to different epitopes        within the binding domain of the natural ligand(s) of said ErbB1        receptor molecule type.    -   (f) A pharmaceutical composition, wherein blocking and/or        inhibition of the ErbB receptor, and induction of        down-regulation of ErbB receptor-specific pathway signaling is        enhanced as compared with a composition comprising a single        antibody molecule which binds to said first or said second        epitope on said ErbB1 receptor molecule type only.    -   (g) A pharmaceutical composition, wherein the induction of        cross-linking and/or dimerization of ErbB receptor molecules of        the same or different specificity is enhanced as compared with a        composition comprising a single antibody molecule which binds to        said first or said second epitope on said ErbB1 receptor        molecule type only.    -   (h) A pharmaceutical composition, wherein said ErbB receptor        molecules, are involved in cross-linking and/or dimerization,        and are selected from ErbB1 and ErbB2 (Her-2).    -   (i) A pharmaceutical composition, wherein said first and/or said        second antibodies is a monospecific antibody.    -   (j) A pharmaceutical composition, wherein the first antibody is        murine, chimeric or humanized MAb 425.    -   (k) A pharmaceutical composition, wherein the second antibody is        murine, chimeric or humanized MAb 225.    -   (l) A pharmaceutical composition, wherein said first antibody is        humanized MAb 425 (h425) and said second antibody is chimeric        MAb 225 (c225).    -   A pharmaceutical composition, wherein said cytotoxic agent is a        chemotherapeutic agent.    -   A pharmaceutical composition, wherein said chemotherapeutic        agent is selected from any of the compounds of the group:        cisplatin, doxorubicin, gemcitabine, docetaxel, paclitaxel,        bleomycin.    -   A pharmaceutical composition, wherein said cytotoxic agent is an        ErbB receptor inhibitor, a VEGF receptor inhibitor, a tyrosine        kinase inhibitor, a protein kinase A inhibitor, an        anti-angiogenic agent, or a cytokine.    -   A pharmaceutical composition, wherein said first and/or said        second antibody molecule is an immunoconjugate, wherein the        antibody portion is fused by its C-terminus to a biologically        effective peptide, polypeptide or protein, optionally via a        linker peptide.    -   A pharmaceutical composition, wherein the protein is a cytokine.    -   A pharmaceutical kit comprising (i) a first package comprising a        first antibody molecule, or a portion thereof, which comprises        binding sites that bind to a first specific epitope present on a        ErbB1 receptor molecule, and (ii) a second package comprising a        second antibody molecule which comprises binding sites that bind        to a second different specific epitope on the same ErbB1        receptor molecule type.    -   A pharmaceutical kit, wherein at least said first or said second        eptitope on the ErbB1 receptor is located within the ErbB1        receptor binding domain.    -   A pharmaceutical kit, wherein said first and said second        eptitope on the ErbB1 receptor is located within the ErbB1        receptor binding domain.    -   A pharmaceutical kit, wherein at least one of said molecules        binds to an epitope within the ErbB1 receptor binding domain to        which the natural ligand of the receptor binds.    -   A pharmaceutical kit, wherein said first antibody molecule is        murine, chimeric or humanized monoclonal antibody 425, and said        second molecule is murine, chimeric or humanized monoclonal        antibody 225.    -   A pharmaceutical kit comprising a first package that comprises        humanized MAb 425 (h425) and a second package that comprises        chimeric MAb 225 (c225).    -   A pharmaceutical kit according to any of the following 6        pharmaceutical kits comprising additionally a third package        comprising a cytotoxic agent.    -   (a) A pharmaceutical kit comprising (i) a first package        comprising a first antibody molecule, or a portion thereof,        which comprises binding sites that bind to a first specific        epitope present on a ErbB1 receptor molecule, and (ii) a second        package comprising a second antibody molecule which comprises        binding sites that bind to a second different specific epitope        on the ErbB1 receptor molecule type.    -   (b) A pharmaceutical kit, wherein at least said first or said        second epitope on the ErbB1 receptor is located within the ErbB1        receptor binding domain.    -   (c) A pharmaceutical kit, wherein said first and said second        epitope on the ErbB1 receptor is located within the ErbB1        receptor binding domain.    -   (d) A pharmaceutical kit, wherein at least one of said molecules        binds to an epitope within the ErbB1 receptor binding domain to        which the natural ligand of the receptor binds.    -   (e) A pharmaceutical kit, wherein said first antibody molecule        is murine, chimeric or humanized monoclonal antibody 425, and        said second molecule is murine, chimeric or humanized monoclonal        antibody 225.    -   (f) A pharmaceutical kit comprising a first package that        comprises humanized MAb 425 (h425) and a second package that        comprises chimeric MAb 225 (c225).    -   A pharmaceutical kit, wherein said cytotoxic agent is a        chemotherapeutic agent.    -   A pharmaceutical kit, wherein said chemotherapeutic agent is        selected from any of the compounds of the group: cisplatin,        doxorubicin, gemcitabine, docetaxel, paclitaxel, bleomycin.    -   A pharmaceutical kit, wherein said cytotoxic drug is an ErbB        receptor inhibitor, a VEGF receptor inhibitor, a tyrosine kinase        inhibitor, a protein kinase A inhibitor, an anti-angiogenic        agent, or a cytokine.    -   Use of a pharmaceutical composition or a pharmaceutical kit as        defined above and in any of the claims, for the manufacture of a        medicament to treat tumors, tumor metastases or tumor related        diseases which are related to overexpression of ErbB, especially        ErbB1 receptors.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the observation that two or more distinctmolecules, preferably monoclonal antibodies (MAbs) with specificitiesfor different immunogenic structures, can bind at the same time andwithout hindrance to their epitopes, which may be located on the sameErbB, preferably ErbB1, receptor domain, e.g. within the ErbB (ErbB1)ligand-binding domain. Application of two or more chemical or biologicalmolecules having the described properties, such as monospecific MAbs orcombinations of antibodies directed against the same or differentreceptors can greatly improve the therapeutic efficacy compared to theefficacy of treatment with only one monospecific antibody:

-   -   Two or more MAbs independently bind to different epitopes on        their target receptor (e.g. EGFR).    -   Due to independent binding to different receptor epitopes the        amount of antibody bound per receptor and thus per cell can be        increased with the same antibody dose or concentration. Under        optimal conditions with saturating concentrations or doses for        each antibody, the number of antibody molecules bound per        receptor and per cell could be theoretically doubled when two        antibodies against different epitopes are used. With every        additionally applied antibody a linear increase of bound        antibody protein per receptor and per cell could be attained.    -   Cells with antigen densities below the threshold for        antibody-dependent immune effector functions, which are not        vulnerable to antibody therapies under normal conditions,        present increased amounts of antibody on their surface after        treatment with two or more antibodies against different epitopes        of the same receptor and thus become potentially accessible for        ADCC and CDC.    -   Compared to the efficiency of receptor blockade obtained with        only one single antibody, application of two or more        monospecific antibodies with specificity for different epitopes        within or near the ligand-binding domain clearly increases        efficacy of receptor blockade.    -   Because receptor blockade by the combination of different        antibodies against the same receptor domain is more effective        than receptor blockade by only one single antibody, a more        effective inhibition of ligand-binding is attained, which        results in a more effective inactivation of the receptor.    -   This more efficient receptor inactivation results in a more        effective inhibition of downstream receptor signaling and        consequently in an increased impact on ligand-dependent cell        functions.    -   Due to the more efficient receptor blockade the dosage (or        concentration) of each of the applied antibodies can be reduced        without loss of efficacy. This can be of great interest when        therapeutic antibodies are applied, which show dose-limiting        toxicities or side effects already below the optimal therapeutic        dose.    -   Monospecific antibodies that react with one single epitope of a        receptor will only allow formation of aggregates consisting of        two receptor molecules. In contrast to this, cross-linking of        receptors induced by two or more antibodies against different        epitopes results in receptor-antibody complexes that contain        clearly larger numbers of receptors.    -   Formation of larger receptor-antibody complexes induced by        application of two or more antibodies improves internalization        of the receptors and thus may be more efficient for removal of        receptors from the cell surface and consequent down-modulation        of receptor-dependent cellular functions.    -   Combinations of two or more antibodies against the same or        different receptors can be used for treatment of tumors carrying        appropriate receptors. EGFR positive tumors are a typical        example, however application of the therapeutic principle        described in this invention is not limited to this indication.        Thus, a wide variety of tumors carrying other receptors,        receptor families or other antigenic structures can be treated        using the same principle.    -   The combined treatment with two or more antibodies directed        against different antigens on the same or different receptors is        also applicable as combination therapy together with        chemotherapeutic drugs and/or irradiation.    -   The combined treatment with two or more antibodies directed        against different antigens on the same or different receptors as        well can be used in combination with other therapeutic        principles including but not limited to treatment with hormone        antagonists or hormone agonists, angiogenesis inhibitors and        other treatments.

The principle of combined treatment with suitable molecules, preferablyantibodies, with different specificities to antigen structures on thesame or different receptors is described here exemplarily for treatmentof EGFR positive tumors. However, this principle is not limited to theEGFR and can be adapted for use with any other target antigen.

If not otherwise pointed out the terms and phrases used in thisinvention have the meanings and definitions as given below. Moreover,these definitions and meanings describe the invention in more detail,preferred embodiments included.

A “receptor” or “receptor molecule” is a soluble or membranebound/associated protein or glycoprotein comprising one or more domainsto which a ligand binds to form a receptor-ligand complex. By bindingthe ligand, which may be an agonist or an antagonist the receptor isactivated or inactivated and may initiate or block pathway signaling.

The term “receptor molecule type” or “ErbB (ErbB1) receptor moleculetype” means a specific receptor type such as ErbB1, ErbB2, etc. but nota specific single molecule of this receptor type. If it is stated hereinthat the antibodies according to the invention within their combinationbind to a specific ErbB receptor molecule type, this does includebinding of the antibodies to the same or different molecules of the sameErbB receptor type as indicated. Thus, it is possible that the firstantibody binds to a specific epitope on an individual ErbB1 receptormolecule, and the second antibody binds to another different epitope ofthe same individual ErbB1 receptor molecule. However, it is alsopossible that the second antibody binds to the same or different epitopeof another individual receptor molecule of the same receptor type.

By “ligand” or “receptor ligand” is meant a natural or syntheticcompound which binds a receptor molecule to form a receptor-ligandcomplex. The term ligand includes agonists, antagonists, and compoundswith partial agonist/antagonist action.

An “agonist” or “receptor agonist” is a natural or synthetic compoundwhich binds the receptor to form a receptor-agonist complex byactivating said receptor and receptor-agonist complex, respectively,initiating a pathway signaling and further biological processes.

By “antagonist” or “receptor antagonist” is meant a natural or syntheticcompound that has a biological effect opposite to that of an agonist. Anantagonist binds the receptor and blocks the action of a receptoragonist by competing with the agonist for receptor. An antagonist isdefined by its ability to block the actions of an agonist. A receptorantagonist may be also an antibody or an immunotherapeutically effectivefragment thereof. Preferred antagonists according to the presentinvention are cited and discussed below.

An “ErbB receptor” is a receptor protein tyrosine kinase which belongs,as already specified above, to the ErbB receptor family and includesEGFR (ErbB1), ErbB2, ErbB3 and ErbB4 receptors and other members of thisfamily to be identified in the future. The ErbB receptor will generallycomprise an extracellular domain, which may bind an ErbB ligand; alipophilic transmembrane domain; a conserved intracellular tyrosinekinase domain; and a carboxyl-terminal signaling domain harboringseveral tyrosine residues which can be phosphorylated. The ErbB receptormay be a “native sequence” ErbB receptor or an “amino acid sequencevariant” thereof. Preferably the ErbB receptor is native sequence humanErbB receptor. ErbB1 refers to the gene encoding the EGFR proteinproduct. Mostly preferred is the EGF receptor (EGFR, HER1). Theexpressions “ErbB1” and “HER1” and “EGFR” are used interchangeablyherein and refer to human HER1 protein. The expressions “ErbB2” and“HER2” are used interchangeably herein and refer to human HER2 protein.ErbB1 receptors (EGFR) are preferred according to this invention

“ErbB ligand” is a polypeptide which binds to and/or activates an ErbBreceptor. ErbB ligands which bind EGFR include, for example, EGF,TGF-alpha, amphiregulin, betacellulin, HB-EGF and epiregulin, preferablyEGF and TGF-alpha.

“ErbB receptor binding domain” is in the context of this invention thelocal region (binding sequence/loop/pocket) of the ErbB receptor towhich a natural ligand or an antagonistic or agonistic drug binds. Thisregion may comprise not only one specific binding site or epitope buttwo or more epitopes and binding sites, respectively. One specificbinding epitope within the domain binds to one kind of antagonistic oragonistic drug or ligand. Nevertheless it is deemed, that the binding ofdifferent agents to different epitopes within or nearly adjacent thebinding domain of the same receptor type generally causes by inhibitionor activation a distinct and unique signaling pathway that is typicalfor said receptor. Moreover, it should be pointed out that the phrase“within the binding domain” used in this description and claims includesalso locations in close vicinity of the real binding domain of therespective natural ligand(s).

“ErbB binding epitope or binding site” means a conformation and/orconfiguration of amino acids within or in close vicinity of the bindingdomain of an ErbB receptor to which ligands or receptorantagonists/agonists bind.

“Same ErbB/ErbB1 receptor molecule” means not necessarily the identicalreceptor molecule, but includes also another receptor molecule of thesame type. Preferably, the term relates to an identical receptormolecule.

The term “ErbB receptor antagonist/inhibitor” refers to a biologicallyeffective molecule, which binds and blocks or inhibits the ErbBreceptor. Thus, by blocking the receptor the antagonist prevents bindingof the ErbB ligand (agonist) and activation of the agonist/ligandreceptor complex. ErbB antagonists may be directed to HER1 (ErbB1,EGFR), HER2 (ErbB2) and ErbB3 and ErbB4. Preferred antagonists of theinvention are directed to the EGF receptor (EGFR, HER1). The ErbBreceptor antagonist may be an antibody or an immunotherapeuticallyeffective fragment thereof or non-immunobiological molecules, such as apeptide, polypeptide protein. Chemical molecules are also included,however, anti-EGFR antibodies and anti-HER2 antibodies are the preferredantagonists according to the invention.

Preferred antibodies of the invention are anti-Her1 and anti-Her2antibodies, more preferably anti-Her1 antibodies. Preferred anti-Her1antibodies are MAb 425, preferably humanized MAb 425 (hMAb 425, U.S.Pat. No. 5,558,864; EP 0531 472) and chimeric MAb 225 (CETUXIMAB®). Mostpreferred is monoclonal antibody h425, which has shown in mono-drugtherapy high efficacy combined with reduced adverse and side effects.Most preferred anti-HER2 antibody is HERCEPTIN® commercialized byGenentech/Roche. Efficacious EGF receptor antagonists according to theinvention may be also natural or synthetic chemical compounds. Someexamples of preferred molecules of this category include organiccompounds, organometallic compounds, salts of organic and organometalliccompounds. Examples for chemical HER2 receptor antagonists are: styrylsubstituted heteroaryl compounds (U.S. Pat. No. 5,656,655); bis monoand/or bicyclic aryl heteroaryl, carbocyclic, and heterocarbocycliccompounds (U.S. Pat. No. 5,646,153); tricyclic pyrimidine compounds(U.S. Pat. No. 5,679,683); quinazoline derivatives having receptortyrosine kinase inhibitory activity (U.S. Pat. No. 5,616,582);heteroarylethenediyl or heteroaryl-ethenediylaryl compounds (U.S. Pat.No. 5,196,446); a compound designated as6-(2,6-dichlorophenyl)-2-(4-(2-diethyl-aminoethoxy)phenylamino)-8-methyl-8H-pyrido(2,3)-5-pyrimidin-7-one(Panek, et al., 1997, J. Pharmacol. Exp. Therap. 283,1433) inhibitingEGFR, PDGFR, and FGFR families of receptors.

The term “tyrosine kinase antagonist/inhibitor” refers according to thisinvention to natural or synthetic agents that are enabled to inhibit orblock tyrosine kinases, receptor tyrosine kinases included. Thus, theterm includes per se ErbB receptor antagonists/inhibitors as definedabove. With exception of the anti-ErbB receptor antibodies mentionedabove and below, more preferable tyrosine kinase antagonist agents underthis definition are chemical compounds which have shown efficacy inmono-drug therapy for breast and prostate cancer. Suitableindolocarbazole-type tyrosine kinase inhibitors can be obtained usinginformation found in documents such as U.S. Pat. Nos. 5,516,771;5,654,427; 5,461,146; 5,650,407. U.S. Pat. Nos. 5,475,110; 5,591,855;5,594,009 and WO 96/11933 disclose pyrrolocarbazole-type tyrosine kinaseinhibitors and prostate cancer. One of the most promising anti-canceragents in this context is gefitinib (IRESSA®, Astra Zeneca), which isreported to possess outstanding therapeutic efficacy and excellenttolerability in patients with non-small cell lung cancer (NSCLC) as wellas advanced head and neck cancer.

Preferably, the dosage of the chemical tyrosine kinase inhibitors asdefined above is from 1 pg/kg to 1 g/kg of body weight per day. Morepreferably, the dosage of tyrosine kinase inhibitors is from 0.01 mg/kgto 100 mg/kg of body weight per day.

The biological molecules according to this invention are preferablyantibodies or fragments thereof or any variations of antibodies such asimmunoconjugates.

In this context, the term “antibody” or “immunoglobulin” herein is usedin the broadest sense and specifically covers intact monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies) formed from at least two intact antibodies, andantibody fragments, so long as they exhibit the desired biologicalactivity. The term generally includes heteroantibodies which arecomposed of two or more antibodies or fragments thereof of differentbinding specificity which are linked together.

Depending on the amino acid sequence of their constant regions, intactantibodies can be assigned to different “antibody (immunoglobulin)classes”. There are five major classes of intact antibodies: IgA, IgD,IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called α, δ, ε, γ and μ respectively.Preferred major class for antibodies according to the invention is IgG,in more detail IgG1 and IgG2.

Antibodies are usually glycoproteins having a molecular weight of about150,000, composed of two identical light (L) chains and two identicalheavy (H) chains. Each light chain is linked to a heavy chain by onecovalent disulfide bond, while the number of disulfide linkages variesamong the heavy chains of different immunoglobulin isotypes. Each heavyand light chain also has regularly spaced intrachain disulfide bridges.Each heavy chain has at one end a variable domain (VH) followed by anumber of constant domains. Each light chain has a variable domain atone end (VL) and a constant domain at its other end. The constant domainof the light chain is aligned with the first constant domain of theheavy chain, and the light-chain variable domain is aligned with thevariable domain of the heavy chain. Particular amino acid residues arebelieved to form an interface between the light chain and heavy chainvariable domains. The “light chains” of antibodies from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. Methods formaking monoclonal antibodies include the hybridoma method described byKohler and Milstein (1975, Nature 256, 495) and in “Monoclonal AntibodyTechnology, The Production and Characterization of Rodent and HumanHybridomas” (1985, Burdon et al., Eds, Laboratory Techniques inBiochemistry and Molecular Biology, Volume 13, Elsevier SciencePublishers, Amsterdam), or may be made by well known recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies mayalso be isolated from phage antibody libraries using the techniquesdescribed in Clackson et al., Nature, 352:624-628 (1991) and Marks etal., J. Mol. Biol., 222:58, 1-597(1991), for example.

The term “chimeric antibody” means antibodies in which a portion of theheavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(e.g.: U.S. Pat. No. 4,816,567; Morrison et al., Proc. Nat. Acad. Sci.USA, 81:6851-6855 (1984)). Methods for making chimeric and humanizedantibodies are also known in the art. For example, methods for makingchimeric antibodies include those described in patents by Boss(Celltech) and by Cabilly (Genentech) (U.S. Pat. Nos. 4,816,397;4,816,567).

“Humanized antibodies” are forms of non-human (e.g., rodent) chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region (CDRs) of the recipient are replaced by residuesfrom a hypervariable region of a non-human species (donor antibody) suchas mouse, rat, rabbit or nonhuman primate having the desiredspecificity, affinity and capacity. In some instances, framework region(FR) residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Methods for making humanized antibodies are described,for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech,U.S. Pat. No. 4,816,397).

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)2, Fv and Fcfragments, diabodies, linear antibodies, single-chain antibodymolecules; and multispecific antibodies formed from antibodyfragment(s). An “intact” antibody is one which comprises anantigen-binding variable region as well as a light chain constant domain(CL) and heavy chain constant domains, CH1, CH2 and CH3. Preferably, theintact antibody has one or more effector functions. Papain digestion ofantibodies produces two identical antigen-binding fragments, called“Fab” fragments, each comprising a single antigen-binding site and a CLand a CH1 region, and a residual “Fc” fragment, whose name reflects itsability to crystallize readily.

The “Fc” region of the antibodies comprises, as a rule, a CH2, CH3 andthe hinge region of an IgG1 or IgG2 antibody major class. The hingeregion is a group of about 15 amino acid residues which combine the CH1region with the CH2-CH3 region.

Pepsin treatment yields an “F(ab′)2” fragment that has twoantigen-binding sites and is still capable of cross-linking antigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions (CDRs) of each variable domain interact to definean antigen-binding site on the surface of the VH-VL dimer. Collectively,the six hypervariable regions confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three hypervariable regions specific for an antigen) hasthe ability to recognize and bind antigen, although at a lower affinitythan the entire binding site.

The “Fab” fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain and has oneantigen-binding site only. “Fab′” fragments differ from Fab fragments bythe addition of a few residues at the carboxy terminus of the heavychain CH1 domain including one or more cysteines from the antibody hingeregion.

F(ab′)2 antibody fragments originally were produced as pairs of Fab′fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known (see e.g. Hermanson,Bioconjugate Techniques, Academic Press, 1996; U.S. Pat. No. 4,342,566).

“Single-chain Fv” or “scFv” antibody fragments comprise the V, and V,domains of antibody, wherein these domains are present in a Singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. Single-chain FVantibodies are known, for example, from Plückthun (The Pharmacology ofMonoclonal Antibodies, Vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994)), WO93/16185; U.S. Pat.Nos. 5,571,894; 5,587,458; Huston et al. (1988, Proc. Natl. Acad. Sci.85, 5879) or Skerra and Plueckthun (1988, Science 240, 1038).

The term “variable” or “FR” refers to the fact that certain portions ofthe variable domains differ extensively in sequence among antibodies andare used in the binding and specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed throughout the variable domains of antibodies. It isconcentrated in three segments called “hypervariable” regions both inthe light chain and the heavy chain variable domains. The more highlyconserved portions of variable domains are called the framework regions(FRs). The variable domains of native heavy and light chains eachcomprise four FRs (FR1-FR4), largely adopting a β-sheet configuration,connected by three hypervariable regions, which form loops connecting,and in some cases forming part of the β-sheet structure. Thehypervariable regions in each chain are held together in close proximityby the FRs and, with the hypervariable regions from the other chain,contribute to the formation of the antigen-binding site of antibodies(see Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991)). The constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody dependent cellularcytotoxicity (ADCC). The term “hypervariable region” or “CDR” when usedherein refers to the amino acid residues of an antibody which areresponsible for antigen-binding. The hypervariable region generallycomprises amino acid residues from a “complementarity determiningregion” or “CDR” (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) inthe light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain; and/or those residues from a“hypervariable loop” (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96(L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol.Biol. 196:901-917 (1987)).

“Framework Region” or “FR” residues are those variable domain residuesother than the hypervariable region residues as herein defined.

The term “monospecific” refers to antibodies according to thisinvention, wherein the two binding sites of the antibody have the samespecificity, thus, being able to bind to the same epitope on thereceptor. Preferably, according to this invention, the pharmaceuticalcompositions comprise monospecific antibodies.

“Bispecific antibodies” (BAbs) are single, divalent antibodies (orimmunotherapeutically effective fragments thereof) which have twodifferently specific antigen binding sites. According to this inventionBAbs are characterized as BAb<MAb 1, MAb 2>, wherein <MAb 1> and <MAb 2>designates the antigen-binding sites deriving from MAb 1 and MAb 2. Forexample the first antigen binding site is directed to an angiogenesisreceptor (e.g. integrin or VEGF receptor), whereas the second antigenbinding site is directed to an ErbB receptor (e.g. EGFR or HER2).Bispecific antibodies can be produced by chemical techniques (see e.g.,Kranz et al. (1981) Proc. Natl. Acad. Sci. USA 78, 5807), by “polydoma”techniques (See U.S. Pat. No. 4,474,893) or by recombinant DNAtechniques, which all are known per se. Further methods are described inWO 91/00360, WO 92/05793 and WO 96/04305. Bispecific antibodies can alsobe prepared from single chain antibodies (see e.g., Huston et al. (1988)Proc. Natl. Acad. Sci. 85, 5879; Skerra and Plueckthun (1988) Science240, 1038). These are analogues of antibody variable regions produced asa single polypeptide chain. To form the bispecific binding agent, thesingle chain antibodies may be coupled together chemically or by geneticengineering methods known in the art. It is also possible to producebispecific antibodies according to this invention by using leucinezipper sequences. The sequences employed are derived from the leucinezipper regions of the transcription factors Fos and Jun (Landschulz etal., 1988, Science 240, 1759; for review, see Maniatis and Abel, 1989,Nature 341, 24). Leucine zippers are specific amino acid sequences about20-40 residues long with leucine typically occurring at every seventhresidue. Such zipper sequences form amphipathic α-helices, with theleucine residues lined up on the hydrophobic side for dimer formation.Peptides corresponding to the leucine zippers of the Fos and Junproteins form heterodimers preferentially (O'Shea et al., 1989, Science245, 646). Zipper containing bispecific antibodies and methods formaking them are also disclosed in WO 92/10209 and WO 93/11162.

The term “fusion protein” refers to a natural or synthetic moleculeconsisting of one ore more biological molecules as defined above,wherein two or more peptide- or protein-based (glycoproteins included)molecules having different specificity are fused together optionally bychemical or amino acid based linker molecules. The linkage may beachieved by C—N fusion or N—C fusion (in 5′→3′ direction), preferablyC—N fusion. Preferred fusion proteins according to the invention are,however, immunoconjugates as defines below.

The term “immunoconjugate” refers to a fusion protein and means anantibody or immunoglobulin, respectively, or a immunologically effectivefragment thereof, which is fused by covalent linkage to anon-immunologically effective molecule. Preferably this fusion partneris a peptide or a protein, which may be glycosylated. Said non-antibodymolecule can be linked to the C-terminal of the constant heavy chains ofthe antibody or to the N-terminals of the variable light and/or heavychains. The fusion partners can be linked via a linker molecule, whichis, as a rule, a 3-15 amino acid residues containing peptide.Immunoconjugates according to this invention are fusion proteinsconsisting of an immunoglobulin or immunotherapeutically effectivefragment thereof, directed to an ErbB receptor, and preferably acytokine, such as TNFα, IFNγ or IL-2, or another toxic agent.Preferably, these peptide- or protein-based molecules are linked withtheir N-terminal to the C-terminal of said immunoglobulin, which is theFc portion thereof.

“Heteroantibodies” are fusion proteins consisting essentially of two ormore antibodies or antibody-binding fragments which are fused togetherby regularly chemical cross-linkers, each of said antibodies having adifferent binding specificity. Heteroantibodies can be prepared byconjugating together two or more antibodies or antibody fragments.Preferred heteroantibodies are comprised of cross-linked Fab/Fab′fragments. A variety of coupling or cross-linking agents can be used toconjugate the antibodies. Examples are protein A, carboiimide,N-succinimidyl-S-acetyl-thioacetate (SATA) andN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (see e.g., Karpovskyet al. (1984) J. EXP. Med. 160, 1686; Liu et a. (1985) Proc. Natl. Acad.Sci. USA 82, 8648). Other methods include those described by Paulus,Behring Inst. Mitt., No. 78, 118 (1985); Brennan et al. (1985) Science30, 81, or Glennie et al. (1987), J. Immunol. 139, 2367. Another methoduses o-phenylenedimaleimide (oPDM) for coupling three Fab′ fragments (WO91/03493). Multispecific antibodies are in context of this inventionalso suitable and can be prepared, for example according to the teachingof WO 94/13804 and WO 98/50431. A preferred heteroantibody according tothis invention is a fusion protein comprising two anti-EGFR antibodies(each antibody is directed to different epitopes of the same receptor)linked together as described (e.g. MAB 425-MAB 225).

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor (VEGF);integrin; thrombopoietin (TPO); nerve growth factors such as NGF.beta.;platelet-growth factor; transforming growth factors (TGFs) such asTGF.alpha. and TGF.beta.; erythropoietin (EPO); interferons such asIFN.alpha., IFN.beta., and IFN.gamma.; colony stimulating factors suchas M-CSF, GM-CSF and G-CSF; interleukins such as IL-1, IL-1a, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; andTNF-.alpha. or TNF-.beta.. Preferred cytokines according to theinvention are interferons, TNF.alpha. and IL-2.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include complement dependent cytotoxicity, Fcreceptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC),phagocytosis; down regulation of cell surface receptors (e.,g. B cellreceptor), etc.

The term “ADCC” (antibody-dependent cell-mediated cytotoxicity) refersto a cell-mediated reaction in which nonspecific cytotoxic cells thatexpress Fc receptors (FcR) (e.g. natural killer (NK) cells, neutrophils,and macrophages) recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. The primary cells formediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. To assess ADCC activity of a moleculeof interest, an in vitro ADCC assay, such as that described in the priorart (U.S. Pat. Nos. 5,500,362; 5,821,337) may be performed. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and natural killer (NK) cells.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcRs are reviewed, for example, inRavetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).

The therapeutic approach of this invention includes as a specificembodiment the administration of further therapeutically effectiveagents, which support the desired effect, e.g. tumor toxicity orcytostatic efficacy, or diminish or prevent undesired side effects. Thusthe invention includes the combination of such agents with thepharmaceutical composition defined and claimed above and below, whereinsaid agents may be other ErbB receptor antagonists, VEGF receptorantagonists, cytokines, cytokine immunoconjugates, anti-angiogenicagents, anti-hormonal agents, or cytotoxic agents in general. It is alsoan object of this invention to combine the compositions as definedherein with radiotherapy according to known methods.

The term “cytotoxic agent” as used in this context is defined verybroadly and refers to a substance that inhibits or prevents the functionof cells and/or causes destruction of cells (cell death), and/or exertsanti-neoplastic/anti-proliferative effects, for example, preventsdirectly or indirectly the development, maturation or spread ofneoplastic tumor cells. The term includes expressively also such agentsthat cause a cytostatic effect only and not a mere cytotoxic effect.

The term includes chemotherapeutic agents as specified below, as well asother ErbB antagonists (such as anti-ErbB antibodies), anti-angiogenicagents, tyrosine kinase inhibitors, protein kinase A inhibitors, membersof the cytokine family, radioactive isotopes, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin.

The term “chemotherapeutic agent” is a subset of the term “cytotoxicagent” and means specifically chemical agents that exert anti-neoplasticeffects, preferably directly on the tumor cell, and less indirectlythrough mechanisms such as biological response modification. Suitablechemotherapeutic agents according to the invention are preferablynatural or synthetic chemical compounds. There are large numbers ofanti-neoplastic chemical agents available in commercial use, in clinicalevaluation and in pre-clinical development, which could be included inthe present invention for treatment of tumors/neoplasia by combinationtherapy with the receptor antagonists as claimed and described in thisinvention. It should be pointed out that the chemotherapeutic agents canbe administered optionally together with said ErbB receptor antagonists,preferably said anti-EGFR antibodies, according to the invention.

Examples of chemotherapeutic or agents include alkylating agents, forexample, nitrogen mustards, ethyleneimine compounds, alkyl sulphonatesand other compounds with an alkylating action such as nitrosoureas,cisplatin and dacarbazine; antimetabolites, for example, folic acid,purine or pyrimidine antagonists; mitotic inhibitors, for example, vincaalkaloids and derivatives of podophyllotoxin; cytotoxic antibiotics andcamptothecin derivatives.

Preferred chemotherapeutic agents are amifostine (ethyol), cisplatin,dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard),streptozocin, cyclophosphamide, carrnustine (BCNU), lomustine (CCNU),doxorubicin (adriamycin), doxorubicin lipo (doxil), gemcitabine(gemzar), daunorubicin, daunorubicin lipo (daunoxome), procarbazine,mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU),vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel(taxotere), aldesleukin, asparaginase, busulfan, carboplatin,cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethyl-camptothecin(SN38), gefitinib (Iressa), dacarbazine, floxuridine, fludarabine,hydroxyurea, ifosfamide, idarubicin, mesna, interferon alpha, interferonbeta, irinotecan, mitoxantrone, topotecan, leuprolide, megestrol,melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase,pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen,teniposide, testolactone, thioguanine, thiotepa, uracil mustard,vinorelbine, chlorambucil and combinations thereof.

Most preferred chemotherapeutic agents according to the invention arecisplatin, gemcitabine, doxorubicin, paclitaxel (taxol) and bleomycin.

An “anti-angiogenic agent” refers to a natural or synthetic compoundwhich blocks, or interferes with to some degree, the development ofblood vessels. The anti-angiogenic molecule may, for instance, be abiological molecule that binds to and blocks an angiogenic growth factoror growth factor receptor. The preferred anti-angiogenic molecule hereinbinds to an receptor, preferably to an integrin receptor or to VEGFreceptor. The term includes according to the invention also a prodrug ofsaid angiogenic agent. The term furthermore includes agents effective asdescribed and also classified as cytotoxic, preferably, chemotherapeuticagents.

There are a lot of molecules having different structure and origin whichelicit anti-agiogenic properties. Most relevant classes of angiogenesisinhibiting or blocking agents which are suitable in this invention, are,for example:

-   -   (i) anti-mitotics such as flurouracil, mytomycin-C, taxol;    -   (ii) estrogen metabolites such as 2-methoxyestradiol;    -   (iii) matrix metalloproteinase (MMP) inhibitors, which inhibit        zinc metalloproteinases metalloproteases) (e.g. betimastat,        BB16, TIMPs, minocycline, GM6001, or those described in        “Inhibition of Matrix Metalloproteinases: Therapeutic        Applications” (Golub, Annals of the New York Academy of Science,        Vol. 878a; Greenwald, Zucker (Eds.), 1999);    -   (iv) anti-angiogenic multi-functional agents and factors such as        IFNα (U.S. Pat. Nos. 4,530,901; 4,503,035; 5,231,176);        angiostatin and plasminogen fragments (e.g. kringle 1-4, kringle        5, kringle 1-3 (O'Reilly, M. S. et al., Cell (Cambridge, Mass.)        79(2): 315-328, 1994; Cao et al., J. Biol. Chem. 271:        29461-29467, 1996; Cao et al., J. Biol. Chem 272: 22924-22928,        1997); endostatin (O'Reilly, M. S. et al., Cell 88(2), 277, 1997        and WO 97/15666), thrombospondin (TSP-1; Frazier, 1991, Curr        Opin Cell Biol 3(5): 792); platelet factor 4 (PF4);    -   (v) plasminogen activator/urokinase inhibitors;    -   (vi) urokinase receptor antagonists;    -   (vii) heparinases;    -   (viii) fumagillin analogs such as TNP-470;    -   (ix) tyrosine kinase inhibitors such as SU1 0. Many of the above        and below mentioned ErbB receptor antagonists (EGFR/HER2        antagonists) are also tyrosine kinase inhibitors, and may show,        therefore anti-EGF receptor blocking activity which results in        inhibiting tumor growth, as well as anti-angiogenic activity        which results in inhibiting the development of blood vessels and        endothelial cells, respectively;    -   (x) suramin and suramin analogs;    -   (xi) angiostatic steroids;    -   (xii) VEGF and bFGF antagonists;    -   (xiii) VEGF receptor antagonists such as anti-VEGF receptor        antibodies (DC-101);    -   (xiv) flk-1 and flt-1 antagonists;    -   (xv) cyclooxxygenase-II inhibitors such as COX-II;    -   (xvi) integrin antagonists and integrin receptor antagonists        such as αv antagonists and αv receptor antagonists, for example,        anti-αv receptor antibodies and RGD peptides. Integrin        (receptor) antagonists are preferred according to this        invention. The term “integrin antagonists/inhibitors” or        “integrin receptor antagonists/inhibitors” refers to a natural        or synthetic molecule that blocks and inhibit an integrin        receptor. In some cases, the term includes antagonists directed        to the ligands of said integrin receptors (such as for α_(v)β₃:        vitronectin, fibrin, fibrinogen, von Willebrand's factor,        thrombospondin, laminin; for α_(v)β₅: vitronectin; for α_(v)β₁:        fibronectin and vitronectin; for α_(v)β₆: fibronectin).

Antagonists directed to the integrin receptors are preferred accordingto the invention. Integrin (receptor) antagonists may be natural orsynthetic peptides, non-peptides, peptidomimetica, immunoglobulins, suchas antibodies or functional fragments thereof, or immunoconjugates(fusion proteins).

Preferred integrin inhibitors of the invention are directed to receptorof α_(v) integrins (e.g. α_(v)β₃, α_(v)β₅, α_(v)β₆ and sub-classes).Preferred integrin inhibitors are α_(v) antagonists, and in particularα_(v)β₃ antagonists. Preferred α_(v) antagonists according to theinvention are RGD peptides, peptidomimetic (non-peptide) antagonists andanti-integrin receptor antibodies such as antibodies blocking α_(v)receptors. Exemplary, non-immunological α_(vβ) ₃ antagonists aredescribed in the teachings of U.S. Pat. Nos. 5,753,230 and 5,766,591.Preferred antagonists are linear and cyclic RGD-containing peptides.Cyclic peptides are, as a rule, more stable and elicit an enhanced serumhalf-life. The most preferred integrin antagonist of the invention is,however, cyclo-(Arg-Gly-Asp-DPhe-NMeVal) (EMD 121974, Cilengitide®,Merck KgaA, Germany; EP 0770 622) which is efficacious in blocking theintegrin receptors α_(v)β₃, α_(v)β₁, α_(v)β₆, α_(v)β₈, α_(IIb)β₃.

Suitable peptidic as well as peptido-mimetic (non-peptide) antagonistsof the α_(v)β₃/α_(v)β₅/α_(v)β₆ integrin receptor have been describedboth in the scientific and patent literature. For example, reference ismade to Hoekstra and Poulter, 1998, Curr. Med. Chem. 5, 195; WO95/32710; WO 95/37655; WO 97/01540; WO 97/37655; WO 97/45137; WO97/41844; WO 98/08840; WO 98/18460; WO 98/18461; WO 98/25892; WO98/31359; WO 98/30542; WO 99/15506; WO 99/15507; WO 99/31061; WO00/06169; EP 0853 084; EP 0854 140; EP 0854 145; U.S. Pat. Nos.5,780,426; and 6,048,861. Patents that disclose benzazepine, as well asrelated benzodiazepine and benzocycloheptene α_(vβ) ₃ integrin receptorantagonists, which are also suitable for the use in this invention,include WO 96/00574, WO 96/00730, WO 96/06087, WO 96/26190, WO 97/24119,WO 97/24122, WO 97/24124, WO 98/15278, WO 99/05107, WO 99/06049, WO99/15170, WO 99/15178, WO 97/34865, WO 97/01540, WO 98/30542, WO99/11626, and WO 99/15508. Other integrin receptor antagonists featuringbackbone conformational ring constraints have been described in WO98/08840; WO 99/30709; WO 99/30713; WO 99/31099; WO 00/09503; U.S. Pat.No. 5,919,792; U.S. Pat. No. 5,925,655; U.S. Pat. No. 5,981,546; andU.S. Pat. No. 6,017,926. In U.S. Pat. No. 6,048,861 and WO 00/72801 aseries of nonanoic acid derivatives which are potent α_(v)β₃ integrinreceptor antagonists were disclosed. Other chemical small moleculeintegrin antagonists (mostly vitronectin antagonists) are described inWO 00/38665. Other α_(v)β₃ receptor antagonists have been shown to beeffective in inhibiting angiogenesis. For example, synthetic receptorantagonists such as(S)-10,11-Dihydro-3-[3-(pyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[a,d]cycloheptene-10-aceticacid (known as SB-265123) have been tested in a variety of mammalianmodel systems. (Keenan et al., 1998, Bioorg. Med. Chem. Lett. 8(22),3171; Ward et al., 1999, Drug Metab. Dispos. 27(11),1232). Assays forthe identification of integrin antagonists suitable for use as anantagonist are described, e.g. by Smith et al., 1990, J. Biol. Chem.265, 12267, and in the referenced patent literature. Anti-integrinreceptor antibodies are also well known. Suitable anti-integrin (e.g.α_(v)β₃, α_(v)β₅, α_(v)β₆) monoclonal antibodies can be modified toencompasses antigen binding fragments thereof, including F(ab)₂, Fab,and engineered Fv or single-chain antibody. One suitable and preferablyused monoclonal antibody directed against integrin receptor α_(v)β₃ isidentified as LM609 (Brooks et al., 1994, Cell 79, 1157; ATCC HB 9537).A potent specific anti-α_(v)β₅ antibody, P1F6, is disclosed in WO97/45447, which is also preferred according to this invention. A furthersuitable α_(v)β₆ selective antibody is MAb 14D9.F8 (WO 99/37683, DSMACC2331, Merck KGaA, Germany) as well as MAb 17.E6 (EP 0719 859, DSMACC2160, Merck KGaA) which is selectively directed to the α_(v)- chainof integrin receptors. Another suitable anti-integrin antibody is thecommercialized Vitraxin®.

As used herein, the term “anti-hormonal agent” includes natural orsynthetic organic or peptidic compounds that act to regulate or inhibithormone action on tumors. In more detail an “anti-hormonal agent” (1)inhibits the production of serum androgens, (2) blocks binding of serumandrogens to androgen receptors, or (3) inhibits the conversion oftestosterone to DHT, or a combination of two or more such compounds. Ananti-hormonal agent according to the invention includes in generalsteroid receptor antagonists and in more detail anti-estrogens includingfor example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, andtoremifene (Fareston); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. The term includes alsoagonists and/or antagonists of glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH) and LHRH (leuteinizing hormone-releasinghormone). A LHRH agonist useful in this invention is goserelin acetate,commercially available as ZOLADEX© (Zeneca). Another example of a usefulLHRH antagonist is GANIRELIX© (Roche/Akzo Nobel). Examples of steroidalanti-androgens are cyproterone acetate (CPA) and megestrol acetate,commercially available as MEGACE® (Bristol-Myers Oncology). Steroidalanti-androgens may block prostatic androgen receptors. They may alsoinhibit the release of LH. CPA is preferably administered to humanpatients at dosages of 100 mg/day to 250 mg/day. Nonsteroidalanti-androgens block androgen receptors. They may also cause an increasein serum LH levels and Serum testosterone levels. A preferrednonsteroidal anti-androgen is flutamide (2-methyl-N-[4-20nitro-3-(trifluoromethyl)phenyll propanamide), commercially available asEULEXIN® (Schering Corp.). Flutamide exerts is anti-androgenic action byinhibiting androgen uptake, by inhibiting nuclear binding of androgen intarget tissues, or both. Another non-steroidal anti-androgen isnilutamide, whose chemical name is5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl-4′-nitrophenyl)-4,4-dimethyl-imidazolidine-dione.In some embodiments of the invention, the anti-hormonal agent is acombination of an LHRH agonist such as leuprolide acetate, and anantiandrogen such as flutamide or nilutamide. For example, leuprolideacetate can be administered by subcucaneous, intramuscular orintravenous injection, and concurrently the flutamide can beadministered orally. Anti-hormonal agents according to the inventioninclude, as pointed out above, antagonists of the steroid/thyroidhormone receptors, including antagonists for other non-permissivereceptors, such as antagonists for RAR, TR, VDR, and the like. Asreadily recognized by those of skill in the art, a variety of retinoicacid receptor (RAR) antagonists, both synthetic and naturally occurring,can be used in accordance with the present invention.

In summary, the pharmaceutical compositions and kits according to thepresent invention preferably can comprise the following drugcombinations:

-   -   (i) Two different monoclonal antibodies (MAb), fragments or        immunoconjugates (preferably immunocytokines) thereof, directed        to different epitopes of the EGF receptor.    -   (ii) MAb 425 and MAb 225 or fragments or immunoconjugates        (preferably immunocytokines) thereof, each directed to different        epitopes of the EGF receptor.    -   (iii) Humanized MAb 425 and chimeric 225 or fragments or        immunoconjugates (preferably immunocytokines) thereof, each        directed to different epitopes of the EGF receptor.    -   (iv) (i) to (iii) in combination with one or more cytotoxic        agents.    -   (v) (i) to (iii), especially MAb 425 and MAb 225 in murine,        chimeric or humanized versions or fragments or immunoconjugates        (preferably immunocytokines) thereof, in combination with one or        more chemotherapeutic agents, preferably cisplatin, gemcitabine        or taxol.    -   (vi) (i) to (iii), especially MAb 425 and MAb 225 in murine,        chimeric or humanized versions or fragments or immunoconjugates        (preferably immunocytokines) thereof, in combination with        another ErbB antagonist.    -   (vii) (i) to (iii), especially MAb 425 and MAb 225 in murine,        chimeric or humanized versions or fragments or immunoconjugates        (preferably immunocytokines) thereof, in combination with an        antibody directed to ErbB-2, preferably Herceptin®, or ErbB-3,        ErbB-4.    -   (viii) (i) to (iii), especially MAb 425 and MAb 225 in murine,        chimeric or humanized versions or fragments or immunoconjugates        (preferably immunocytokines) thereof, in combination with drugs        selected from the following group:        -   tyrosine kinase inhibitors, such as Iressa®;        -   anti-angiogenic agents, preferably integrin inhibitors, more            preferably RGD peptides, cyclic peptides included, such as            cyclo-(Arg-Gly-Asp-DPhe-NMeVal) (Cilengitide®, Merck KGaA);        -   anti-VEGF receptor antibodies, such as DC-101, or VEGF            antagonists;        -   COX-II inhibitors;        -   cytokines, such as TNF-α, IFN-α, IFN-β, IFN-γ, IL-2;        -   type I protein kinase A (PKAI) inhibitors, such as mixed            backbone antisense oligonucleotides, like HYB 165 (see, for            example, Tortora et al., 1999, Clin. Cancer Res., 875-881);        -   anti-hormonal agents, such as goserelin, boserelin,            leuprorelin, tamoxifen.

The terms “cancer” and “tumor” refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. By means of the pharmaceutical compositions according of thepresent invention tumors can be treated such as tumors of the breast,heart, lung, small intestine, colon, spleen, kidney, bladder, head andneck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood,thymus, uterus, testicles, cervix, and liver. More specifically thetumor is selected from the group consisting of adenoma, angio-sarcoma,astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma,hamartoma, hemangioendothelioma, hemangiosarcoma, hematoma,hepato-blastoma, leukemia, lymphoma, medulloblastoma, melanoma,neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcomaand teratoma. In detail, the tumor is selected from the group consistingof acral lentiginous melanoma, actinic keratoses, adenocarcinoma,adenoid cycstic carcinoma, adenomas, adenosarcoma, adenosquamouscarcinoma, astrocytic tumors, bartholin gland carcinoma, basal cellcarcinoma, bronchial gland carcinomas, capillary, carcinoids, carcinoma,carcinosarcoma, cavernous, cholangio-carcinoma, chondosarcoma, choriodplexus papilloma/carcinoma, clear cell carcinoma, cystadenoma,endodermal sinus tumor, endometrial hyperplasia, endometrial stromalsarcoma, endometrioid adenocarcinoma, ependymal, epitheloid, Ewing'ssarcoma, fibrolamellar, focal nodular hyperplasia, gastrinoma, germ celltumors, glioblastoma, glucagonoma, hemangiblastomas,hemangioendothelioma, hemangiomas, hepatic adenoma, hepaticadenomatosis, hepatocellular carcinoma, insulinoma, intraepithelialneoplasia, interepithelial squamous cell neoplasia, invasive squamouscell carcinoma, large cell carcinoma, leiomyosarcoma, lentigo malignamelanomas, malignant melanoma, malignant mesothelial tumors,medulloblastoma, medulloepithelioma, melanoma, meningeal, mesothelial,metastatic carcinoma, mucoepidermoid carcinoma, neuroblastoma,neuroepithelial adenocarcinoma nodular melanoma, oat cell carcinoma,oligodendroglial, osteosarcoma, pancreatic polypeptide, papillary serousadeno-carcinoma, pineal cell, pituitary tumors, plasmacytoma,pseudo-sarcoma, pulmonary blastoma, renal cell carcinoma,retinoblastoma, rhabdomyo-sarcoma, sarcoma, serous carcinoma, small cellcarcinoma, soft tissue carcinomas, somatostatin-secreting tumor,squamous carcinoma, squamous cell carcinoma, submesothelial, superficialspreading melanoma, undifferentiated carcinoma, uveal melanoma,vermucous carcinoma, vipoma, well differentiated carcinoma, and Wilm'stumor.

Tumors which can be preferably be treated with the antibody moleculesaccording to the invention are solid tumors or tumor metastases thatexpress ErbB receptors, especially ErbB1 receptors, in high amounts,such as breast cancer, prostate cancer head and neck cancer, SCLC,pancreas cancer.

The term “biologically/functionally effective” or “therapeuticallyeffective (amount)” refers to a drug/molecule which causes a biologicalfunction or a change of a biological function in vivo or in vitro, andwhich is effective in a specific amount to treat a disease or disorderin a mammal, preferably in a human. In the case of cancer, thetherapeutically effective amount of the drug may reduce the number ofcancer cells; reduce the tumor size; inhibit (i.e., slow to some extentand preferably stop) cancer cell infiltration into peripheral organs;inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Tothe extent the drug may prevent growth and/or kill existing cancercells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can, for example, be measured by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

The term “immunotherapeutically effective” refers to biologicalmolecules which cause an immune response in a mammal. More specifically,the term refers to molecules which may recognize and bind an antigen.Typically, antibodies, antibody fragments and antibody fusion proteinscomprising their antigen binding sites (complementary determiningregions, CDRs) are immunotherapeutically effective.

“Radiotherapy”: According to the invention the tumors can additionallybe treated with radiation or radiopharmaceuticals. The source ofradiation can be either external or internal to the patient beingtreated. When the source is external to the patient, the therapy isknown as external beam radiation therapy (EBRT). When the source ofradiation is internal to the patient, the treatment is calledbrachytherapy (BT). Some typical radioactive atoms that have been usedinclude radium, cesium-137, and iridium-192, americium-241 and gold-198,Cobalt-57; Copper-67; Technetium-99; Iodide-123; Iodide-131; andIndium-111. It is also possible to label the agents according to theinvention with radioactive isotopes. Today radiation therapy is thestandard treatment to control unresectable or inoperable tumors and/ortumor metastases. Improved results have been seen when radiation therapyhas been combined with chemotherapy. Radiation therapy is based on theprinciple that high-dose radiation delivered to a target area willresult in the death of reproductive cells in both tumor and normaltissues. The radiation dosage regimen is generally defined in terms ofradiation absorbed dose (rad), time and fractionation, and must becarefully defined by the oncologist. The amount of radiation a patientreceives will depend on various consideration but the two most importantconsiderations are the location of the tumor in relation to othercritical structures or organs of the body, and the extent to which thetumor has spread. A preferred course of treatment for a patientundergoing radiation therapy will be a treatment schedule over a 5 to 6week period, with a total dose of 50 to 60 Gy administered to thepatient in a single daily fraction of 1.8 to 2.0 Gy, 5 days a week. A Gyis an abbreviation for Gray and refers to a dose of 100 rad. If tumorsare treated with the anti-ErbB antibodies as described in this inventionin context with a radiation regimen, usually a positive and evensynergistic effect can be observed. In other words, the inhibition oftumor growth by means of said compounds is enhanced when combined withradiation and/or chemotherapeutic agents. Radiation therapy can beoptionally used according to the invention. It is recommended andpreferred in cases in which no sufficient amounts of the agentsaccording to the invention can be administered to the patient.

“Pharmaceutical treatment”: The method of the invention comprises avariety of modalities for practicing the invention in terms of thesteps. For example, the agents according to the invention can beadministered simultaneously, sequentially, or separately. Furthermore,the agents can be separately administered within a time interval ofabout 3 weeks between administrations, i.e., from substantiallyimmediately after the first active agent is administered to up to about3 weeks after the first agent is administered. The method can bepracticed following a surgical procedure. Alternatively, the surgicalprocedure can be practiced during the interval between administration ofthe first active agent and the second active agent. Exemplary of thismethod is the combination of the present method with surgical tumorremoval. Treatment according to the method will typically compriseadministration of the therapeutic compositions in one or more cycles ofadministration. For example, where a simultaneous administration ispracticed, a therapeutic composition comprising both agents isadministered over a time period of from about 2 days to about 3 weeks ina single cycle. Thereafter, the treatment cycle can be repeated asneeded according to the judgment of the practicing physician. Similarly,where a sequential application is contemplated, the administration timefor each individual therapeutic will be adjusted to typically cover thesame time period. The interval between cycles can vary from about zeroto 2 months.

The agents of this invention can be administered parenterally byinjection or by gradual infusion over time. Although the tissue to betreated can typically be accessed in the body by systemic administrationand therefore most often treated by intravenous administration oftherapeutic compositions, other tissues and delivery means arecontemplated where there is a likelihood that the tissue targetedcontains the target molecule. Thus, the agents of this invention can beadministered intraocularly, intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, transdermally, byorthotopic injection and infusion, and can also be delivered byperistaltic means. The therapeutic compositions containing, for example,an integrin antagonist of this invention are conventionally administeredintravenously, as by injection of a unit dose, for example. Therapeuticcompositions of the present invention contain a physiologicallytolerable carrier together with the relevant agent as described herein,dissolved or dispersed therein as an active ingredient.

As used herein, the term “pharmaceutically acceptable” refers tocompositions, carriers, diluents and reagents which represent materialsthat are capable of administration to or upon a mammal without theproduction of undesirable physiological effects such as nausea,dizziness, gastric upset and the like. The preparation of apharmacological composition that contains active ingredients dissolvedor dispersed therein is well understood in the art and need not belimited based on formulation. Typically, such compositions are preparedas injectables either as liquid solutions or suspensions, however, solidforms suitable for solution, or suspensions, in liquid prior to use canalso be prepared. The preparation can also be emulsified. The activeingredient can be mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient and in amountssuitable for use in the therapeutic methods described herein. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanolor the like and combinations thereof. In addition, if desired, thecomposition can contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents and the like whichenhance the effectiveness of the active ingredient. The therapeuticcomposition of the present invention can include pharmaceuticallyacceptable salts of the components therein. Pharmaceutically acceptablesalts include the acid addition salts (formed with the free amino groupsof the polypeptide) that are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, tartaric, mandelic and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like. Particularly preferred is theHCl salt when used in the preparation of cyclic polypeptide αvantagonists. Physiologically tolerable carriers are well known in theart. Exemplary of liquid carriers are sterile aqueous solutions thatcontain no materials in addition to the active ingredients and water, orcontain a buffer such as sodium phosphate at physiological pH value,physiological saline or both, such as phosphate-buffered saline. Stillfurther, aqueous carriers can contain more than one buffer salt, as wellas salts such as sodium and potassium chlorides, dextrose, polyethyleneglycol and other solutes. Liquid compositions can also contain liquidphases in addition to and to the exclusion of water. Exemplary of suchadditional liquid phases are glycerin. vegetable oils such as cottonseedoil, and water-oil emulsions.

Typically, a therapeutically effective amount of an immunotherapeuticagent, for example, in the form of an ErbB (ErbB1) receptor blockingantibody, an integrin receptor blocking antibody or antibody fragment orantibody conjugate or an anti-VEGF receptor blocking antibody, fragmentor conjugate is an amount such that, when administered inphysiologically tolerable composition, is sufficient to achieve a plasmaconcentration of from about 0.01 microgram (μg) per milliliter (ml) toabout 100 μg/ml, preferably from about 1 μg/ml to about 5 μg/ml andusually about 5 μg/ml. Stated differently. the dosage can vary fromabout 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg toabout 200 mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg,in one or more dose administrations daily for one or several days. Wherethe immunotherapeutic agent is in the form of a fragment of a monoclonalantibody or a conjugate, the amount can readily be adjusted based on themass of the fragment/conjugate relative to the mass of the wholeantibody. A preferred plasma concentration in molarity is from about 2micromolar (μM) to about 5 millimolar (mM) and preferably, about 100 μMto 1 mM antibody antagonist.

A therapeutically effective amount of an agent according of thisinvention which is a non-immunotherapeutic peptide or a proteinpolypeptide or other similarly-sized biological molecule, is typicallyan amount of polypeptide such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 0.1 microgram (μg) per milliliter (ml) toabout 200 μg/ml, preferably from about 1 μg/ml to about 150 μg/ml. Basedon a polypeptide having a mass of about 500 grams per mole, thepreferred plasma concentration in molarity is from about 2 micromolar(μM) to about 5 millimolar (mM) and preferably about 100 μM to 1 mMpolypeptide antagonist.

The typical dosage of an active agent, which is a preferably a chemicalcytotoxic or chemotherapeutic agent according to the invention (neitheran immunotherapeutic agent nor a non-immunotherapeutic peptide/protein)is 10 mg to 1000 mg, preferably about 20 to 200 mg, and more preferably50 to 100 mg per kilogram body weight per day. The pharmaceuticalcompositions of the invention can comprise phrase encompasses treatmentof a subject with agents that reduce or avoid side effects associatedwith the combination therapy of the present invention (“adjunctivetherapy”), including, but not limited to, those agents, for example,that reduce the toxic effect of anticancer drugs, e.g., bone resorptioninhibitors, cardioprotective agents. Said adjunctive agents prevent orreduce the incidence of nausea and vomiting associated withchemotherapy, radiotherapy or operation, or reduce the incidence ofinfection associated with the administration of myelosuppressiveanticancer drugs. Adjunctive agents are well known in the art. Theimmunotherapeutic agents according to the invention can additionallyadministered with adjuvants like BCG and immune system stimulators.Furthermore, the compositions may include immunotherapeutic agents orchemotherapeutic agents which contain cytotoxic effective radio-labeledisotopes, or other cytotoxic agents, such as a cytotoxic peptides (e.g.cytokines) or cytotoxic drugs and the like.

The term “pharmaceutical kit” for treating tumors or tumor metastasesrefers to a package and, as a rule, instructions for using the reagentsin methods to treat tumors and tumor metastases. A reagent in a kit ofthis invention is typically formulated as a therapeutic composition asdescribed herein, and therefore can be in any of a variety of formssuitable for distribution in a kit. Such forms can include a liquid,powder, tablet, suspension and the like formulation for providing thepharmaceutical molecules of this invention, preferably the anti-ErbB1antibodies. The reagents may be provided in separate containers suitablefor administration separately according to the present methods, oralternatively may be provided combined in a composition in a singlecontainer in the package. The package may contain an amount sufficientfor one or more dosages of reagents according to the treatment methodsdescribed herein. A kit of this invention also contains “instructionsfor use” of the materials contained in the package.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1 describes the binding of MAb 425 (EMD 72000) and c225 (Cetuximab)to different cancer cells (A431, SK-OV3, HCT 116, MiaPaca-2, KYSE-30,KYSE-70) alone and in combination, measured by flow cytometry.

FIG. 2 depicts effector target cell aggregation in dependency on theantibody concentration (MAb 425, MAb 225, or a mixture of both).

FIG. 3 shows the inhibition of EGF binding to A431 tumor cells by MAb425, MAb 225, or a mixture of both.

FIG. 4 depicts the displacement of bound EGF on A431 cancer cells by MAb425, MAb 225, or a mixture of both.

FIG. 5 shows the down-modulation of EGF receptor on A431 cells by amixture of humanized MAb 425 (EMD 72000) and chimeric MAb 225.

EXAMPLES Example 1

Increased Antibody Binding on a Per Cell Basis by Combining TwoEGFR-Blocking Antibodies (Cetuximab, EMD 72 000) With Different EpitopeSpecificities.

Six EGFR positive human tumor cell lines originating from epidermoidcarcinoma of the vulva (A431), adenocarcinoma of the ovary (SK-OV-3),colon carcinoma (HCT 116), pancreas carcinoma (MiaPaca-2) and esophaguscarcinoma (KYSE-30, KYSE-70) expressing different EGFR levels wereincubated for 15 minutes on ice with 10 μg/ml Cetuximab or EMD 72 000,respectively, or with a mixture containing finally 2.5 μg/ml Cetuximaband 2.5 μg/ml EMD 72 000 (total MAb concentration: 5 μg/ml). Thereaftercells were washed and incubated for additional 15 minutes on ice with 20μg/ml FITC labeled goat anti-human IgG+IgM(H+L)-F(ab′)₂ as 2nd stepreagent. After washing cells were analyzed by flow cytometry (FACScan.Becton Dickinson) for their fluorescence intensities, which are roughlyequivalent to the amount of antibody bound per cell. As shown,independent of the reduced staining concentration used for the antibodymixture, fluorescence intensities of cells stained with this mixturewere in all cases greater than fluorescence intensities of cells thatwere stained only with one of both antibodies at a higher concentration(FIG. 1).

Example 2

Effector-Target Cell Aggregation as Prerequisite for Antibody-DependentCell-Mediated Cytotoxicity and its Improvement by Combination of TwoAntibodies with Specificity for Different Epitopes of the Human EGFR(Cetuximab and EMD 72 000).

In this model experiment EGFR positive A431 cells were used as targetcells. EGFR negative, Fc-gamma receptor (CD64 (FcγRI) and CD32 (FcγRII))positive U937 histiocytic lymphoma cells were used to mimic effectorcells. A431 cells were fluorescence labeled with the green PKH2, U937cells with red PKH26. Thereafter both cell lines were mixed with aneffector-target cell ratio of 3:1 and incubated for 15 minutes on icewith serial dilutions (final concentrations: 4.74−0.0015μg/ml=3.16×10⁻⁸−1×10⁻¹¹ M as calculated with a MW of 150 kDa for bothantibodies) Cetuximab and EMD 72 000, respectively, or with a serialdilution of a mixture of both antibodies containing half the totalimmunoglobulin concentration (2.37−0.00075 μg/ml=1.58×10⁻⁸−5×10⁻¹² M;the concentration of each of the MAbs in this mixture were ½ of thisconcentration). After centrifugation for 5 minutes at 50×g and 4° C.cells were incubated for further 60 minutes on ice without destroyingthe pellets. Finally pellets were carefully resuspended and proportionsof aggregates determined by flow cytometry using a FACScan instrument.

As shown in FIG. 2, compared to results from incubation of cells withonly one single antibody, the maximum percentage of aggregates isincreased in samples, which were incubated with a mixture of both MAbsat a lower total protein concentration. The increasing part of thetitration curve for the antibody mixture is not significantly displaceddespite of the reduced protein concentration in these samples comparedto samples incubated with only one of the MAbs.

Example 3

Enhanced Inhibition of EGF Binding to A431 Tumor Cells by a Mixture ofTwo Antibodies Directed Against Different Epitopes of the EGFRLigand-Binding Domain.

A431 cells were preincubated for 15 minutes on ice with 0.5 μg/ml EMD 72000, Cetuximab or a mixture of both antibodies at the sameconcentration. After washing off unbound antibody cells were incubatedfor another 15 minutes on ice with 0.01, 0.1, 1 or 10 μg/ml FITC-labeledEGF from mouse submaxillary glands (Molecular Probes Europe, Leiden, TheNetherlands), washed and analyzed by flow cytometry.

Both antibodies strongly inhibited binding of EGF-FITC at allconcentrations. The mixture of both antibodies however was moreeffective than either antibody alone in all cases (FIG. 3).

Example 4

Displacement Bound EGF from EGFR of A431 Cells.

A431 cells were preincubated for 15 minutes on ice with 10 μg/mlFITC-labeled EGF from mouse submaxillary glands (Molecular ProbesEurope, Leiden, The Netherlands). Thereafter cells were washed andincubated for 15 minutes with 10, 1 or 0.1 μg/ml of EMD 72 000 orCetuximab, respectively, or with a mixture of 2.5, 0.25 or 0.025 μg/mlof both antibodies (total immunoglobulin concentration: 5, 0.5 or 0.05μg/ml). Cells were then washed and analyzed by flow cytometry for boundEGF-FITC.

Both antibodies as single agents in a concentration-dependent mannerdisplaced EGF from EGFR of A431 cells. The mixture of both antibodies,which contained only one fourth of the antibody concentrations (½ oftotal immunoglobulin) of each MAb, was similarly effective indisplacement of the FITC-labeled ligand than each of both antibodies ata higher concentration.

Example 5

Down-Modulation of EGFR by a Mixture of at Least Two Antibodies AgainstDifferent Receptor Epitopes but No or Only Minor Reduction of CellSurface EGFR Levels by One Single Antibody After a 24-Hours Incubationof A431 Cells with MAbs.

2×10⁶ A431 cells in 2.5 ml medium containing 10% FCS (fetal calf serum)were seeded into wells of 6-well microplates. Antibodies (Cetuximab, EMD72 000) were added to the cultures at a final concentration of 10 μg/ml.Mixtures of both antibodies were used at final concentrations for eachantibody of 10 μg/ml (mixture 1) or 5 μg/ml (mixture 2), resulting in atotal antibody concentration of 20 and 10 μg/ml respectively. Cells werethen incubated in the presence of the antibodies for 24 hours at 37° C.and 10% CO₂. Thereafter cells were harvested by Trypsin/EDTA(0.05/0.02%) treatment, washed and incubated for 15 minutes on ice witheither 20 μg/ml FITC-labeled goat anti-human IgG+IgM(H+L)-F(ab′)₂ as 2ndstep reagent for detection of surface-bound anti-EGFR antibodies or 10μg/ml of FITC-labeled MAb425 (murine predecessor of EMD 72 000) fordetection of free, non-occupied binding-sites for EMD 72 000. Finallycells were analyzed by flow cytometry. FIG. 5 demonstrates that EMD 72000 and its murine predecessor MAb425 compete for binding to theirepitopes on EGFR and that pre-bound EMD 72 000 nearly completely caninhibit binding of MAb425-FITC. In contrast to this, pre-bound Cetuximabonly minimally inhibits binding of MAb425-FITC compared to untreatedcontrol cells. This clearly indicates that both antibodies bind todistinct epitopes of EGFR. Furthermore this figure shows that afterincubation of cells for 24 hours in presence of both concentrations ofthe antibody mixture the fluorescence intensity of FITC-labeled 2nd stepreagent used for detection of surface-bound antibodies is clearlyreduced.

This indicates that surface EGFR levels of cells treated with theantibody mixture are clearly reduced compared to cells that werecultured with only one of the antibodies. Thus, larger receptor-antibodycomplexes formed by the mixture of MAbs seem to be internalized and/orprocessed by other mechanisms than small receptor-antibody complexesconsisting of only two receptors after cross-linking by one antibodymolecule.

1. A pharmaceutical composition comprising a first antibody or a binding fragment thereof and a second antibody or a binding fragment thereof, having the capability to bind to different epitopes located on an ErbB1 receptor molecule, wherein said first antibody molecule or a portion thereof comprises a binding site that bind to a first specific epitope on the ErbB1 receptor molecule, and said second antibody molecule comprises a binding site that binds to a second specific different epitope on said ErbB1 receptor molecule, wherein said first and said second epitopes on the ErbB1 receptor molecule are located within the ErbB1 receptor ligand-binding domain, thereby increasing the amount of antibody bound per receptor and per cell by the same antibody dose and causing an enhanced ErbB1 blocking or inhibition and induction of down-regulation of ErbB1 receptor-specific pathway signaling as compared with a composition comprising said first or second antibody molecule only, wherein said first antibody is humanized MAb 425 (h425) and said second antibody is chimeric MAb 225 (c225).
 2. The pharmaceutical composition according to claim 1, wherein said receptor ligand-binding domain is the binding domain of the natural ligand of said ErbB1 receptor.
 3. The pharmaceutical composition according to claim 1, wherein said composition exhibits enhanced induction of cross-linking and/or dimerization of ErbB1 receptor molecule compared to a composition comprising a single antibody molecule which binds to said first or said second epitope on said ErbB1 receptor molecule.
 4. The pharmaceutical composition according to claim 1, further comprising a cytotoxic agent.
 5. The pharmaceutical composition according to claim 4, wherein said cytotoxic agent is a chemotherapeutic agent.
 6. The pharmaceutical composition according of claim 5, wherein said chemotherapeutic agent is cisplatin, doxorubicin, gemcitabine, docetaxel, paclitaxel, or bleomycin.
 7. The pharmaceutical composition according of claim 4, wherein said cytotoxic agent is an ErbB receptor inhibitor, a VEGF receptor inhibitor, a tyrosine kinase inhibitor, a protein kinase A inhibitor, an anti-angiogenic agent, or a cytokine.
 8. A kit comprising the pharmaceutical composition of claim 1 in one package and a carrier in a second package.
 9. A method of inhibiting tumor growth and/or metastasis comprising contacting said tumor with the pharmaceutical composition according of claim
 1. 10. The method according to claim 9, wherein the tumor is breast cancer, prostate cancer, head and neck cancer, SCLC or pancreas cancer. 